BR112015015050B1 - Method for diagnosing melanoma in an individual, and, method for determining probability of responding to treatment with a kinase inhibitor in an individual with melanoma - Google Patents

Abstract

METHODS FOR DIAGNOSING MELANOMA AND BASAL CELL CARCINOMA IN AN INDIVIDUAL AND FOR DETERMINING LIKELIHOOD OF RESPONDING TO TREATMENT WITH A KINASE INHIBITOR IN AN INDIVIDUAL WITH MELANOMA OR BASAL CELL CARCINOMA, AND, USE OF A DDR2 INHIBITOR. Methods for diagnosing melanoma or basal cell carcinoma based on mutations in the DDR2 gene are described here. Additionally, a distinct subgroup of BRAF-mutated melanomas also has somatic mutations in the DDR2 gene. Applications of this discovery to routine diagnostics include molecular stratification of melanoma, and tissue identification of targetable DDR2 kinase mutations in routine formalin-fixed paraffin-embedded sections. Methods, compositions and kits related to the discovery that DDR2 mutations can be markers for melanoma in general, and BRAF-mediated melanoma in particular, are described, opening the possibility of dual therapy for melanoma targeting both DDR2 and BRAF.

Description

FIELD OF INVENTION

[001] The present technology refers to novel mutations in the DDR2 gene in melanoma and basal cell carcinoma.

BASICS OF THE INVENTION

[002] The following description is provided to aid the reader's understanding. None of the information provided or references cited is considered to be prior art of the present invention.

Melanoma and Basal Cell Carcinoma

[003] Skin cancer is the most common of all cancers, afflicting more than a million Americans each year, a number that is rising rapidly. It is also the easiest to cure, if diagnosed and treated early. If allowed to progress to the point where it spreads to other locations, the prognosis is very poor. More than 8,000 deaths from melanoma currently occur each year.

[004] Melanoma is a malignant tumor of melanocytes. Melanocytes occur predominantly in the skin, between the outer layer of the skin (the epidermis) and the next layer (the dermis), but are also found in other parts of the body, including the intestine and the eye (see uveal melanoma). Melanoma can occur in any part of the body that contains melanocytes or as a metastatic tumor of unknown primary lesion. Melanoma is less common than other skin cancers, but it is much more dangerous and causes the majority (75%) of skin cancer-related deaths.

[005] Melanoma arises from DNA damage in melanocytes. The early stage of the disease typically begins with a radial growth phase, when the tumor is confined to the epidermis, followed by a dermal “vertical growth phase” (VGP). Some melanomas reach even more invasive potential, growing into neighboring tissue and can spread around the body through the blood or lymphatic vessels to form metastases.

[006] An immunological reaction against the tumor during VGP can be judged by the presence and activity of tumor-infiltrating lymphocytes (TILs). These cells sometimes attack the primary tumor and, in certain cases, the primary tumor regresses with a diagnosis of only the metastatic tumor.

[007] Multiple genetic events have been related to the pathogenesis (development of the disease) of melanoma. Some cases of melanoma have a clear genetic predisposition. Germline mutations in CDKN2A, CDK4, MC1R, MDM2 SNP309, and genes associated with xeroderma pigmentosa (XP) predispose patients to develop melanoma. Other cases of familial melanoma are genetically heterogeneous, and putative loci for familial melanoma have been identified on chromosome arms 1p, 9p, and 12q.

Clinical and Pathological Diagnosis

[008] Melanoma is typically first detected by visual examination of pigmented skin lesions, notably those that show: (A) asymmetry, (B) a border that is irregular, serrated or notched, (C) coloring different shades of brown, black or tan and (D) diameter that has recently changed size. On the contrary, non-neoplastic warts or moles are symmetrical, have a regular border, uniform color and do not show any change in size/diameter over time. The main diagnostic concern is distinguishing a benign mole, a dysplastic mole, which may show progression over time, and a melanoma. Warts that are irregular in color or shape undergo further development into melanoma. After a visual examination and a dermoscopic examination, or in vivo diagnostic tools such as a confocal microscope, a sample (biopsy) of the suspected wart is usually obtained.

Sample Preparation

[009] When an atypical wart is identified, a skin biopsy takes place in order to better diagnose it. Local anesthetic is used to numb the area, then the wart is biopsied. The biopsy material is then sent to a laboratory to be evaluated by a pathologist. A skin biopsy can be a punch or scrape biopsy, or complete excision. Complete excision is the preferred method, but a punch biopsy may suffice if the patient has cosmetic concerns (i.e., the patient does not want a scar) and the lesion is small. A curette or deep shave biopsy is generally avoided due to the risk of transecting a melanoma and thereby losing important prognostic information.

[0010] Most dermatologists and dermatopathologists use a diagnostic scheme to classify melanocytic lesions based on how symmetrical the lesion is and the degree of cytological atypia in the melanocytes. In this classification, a mole is classified as unequivocally benign, atypical/dysplastic, or clearly melanoma. A benign mole exhibits no cytological atypia and significant symmetrical growth. An atypical wart is interpreted as either symmetrical growth and/or cytological atypia (medium, moderate or severe). Typically, cytologic atypia is of greater clinical concern than architectural atypia. Along with melanoma, moles with moderate to severe cytological atypia may require additional excision to ensure that the surgical margin is completely free of the lesion.

[0011] Important aspects of the skin biopsy report melanoma, including the pattern (presence/absence of an in situ component, radial or vertical growth), depth of invasion, presence of lymphocyte infiltrate, presence/absence of vascular or lymphatic invasion, presence/absence of a pre-existing benign melanoma and the mitotic index. An additional important aspect of skin biopsy reports of atypical moles and melanoma is for the pathologist to indicate whether the excision margin is free of tumor. If there are any atypical melanocytes at the margin, or if a melanoma is diagnosed, a re-excision is performed. Lymph node dissection may also be performed based on tumor parameters seen on initial biopsy and reexcision.

[0012] Additional molecular testing can be performed on melanoma, re-excision, or metastatic lymph node biopsy samples to evaluate for targetable genetic changes to help select optimal therapy.

BRAF

[0013] BRAF is a human gene that produces a protein called B-Raf. The gene is also referred to as B-Raf proto-oncogene and B1 homolog of the v-Raf murine viral sarcoma oncogene, while the protein is more formally known as B-Raf serine/threonine protein kinase. B-Raf is a member of the Raf kinase family of specific serine/threonine protein kinases. This protein plays a role in regulating the MAP/ERK kinase signaling pathway, which affects cell division, differentiation, and growth factor expression.

[0014] In 2002, BRAF was shown to be mutated in human cancers. More than 30 BRAF gene mutations associated with human cancers have been identified. The frequency of BRAF mutations varies widely in human cancers from approximately 60% of melanomas and some types of benign moles, to approximately 1-10% of common carcinomas such as lung adenocarcinoma (ACA) and colorectal cancer. In 90% of BRAF mutated tumors, thymine is used to replace adenine at nucleotide 1799. This leads to valine (V) being used to replace glutamate (E) at codon 600 (V600E) in the activation segment. This mutation has been widely observed in papillary thyroid carcinoma, colorectal cancer, melanoma, and non-small cell lung cancer. In June 2011, a team of Italian scientists used massively parallel sequencing to identify the V600E mutation as a likely driver mutation in 100% of hair cell leukemia cases. Less commonly, V600E mutation can also occur due to a double nucleotide substitution.

[0015] BRAF mutations that were found are R462I, I463S, G464E, G464V, G466A, G466E, G466V, G469A, G469E, N581S, E586K, D594V, F595L, G596R, L597V, T599I, V600D, V600E, V 600K, V600R, K601E, E602K and A728V, etc. Most of these mutations are clustered in two regions of the gene: the glycine-rich P loop of the N lobe and the activation segment and flanking regions. Many of these mutations change the activation segment from an inactive state to an active state. For example, in V600 mutations, the aliphatic side chain of Val600 interacts with the phenyl ring of Phe467 in the P loop. Replacing the mid-synthesized hydrophobic Val side chain with a larger, charged residue (such as changes of Val to Glu, Asp , Lys, or Arg seen in human tumors) can destabilize the interactions that maintain the DFG motif in an inactive conformation, resulting in a conformational shift in the active position. Each BRAF kinase mutation has a variable effect on MEK phosphorylation activity, with most mutations having greater phosphorylation activity than the unmutated B-Raf protein, but some mutations showing lower kinase activity, or even no activity, called "mutations" BRAF inhibitors. The effect of these inhibitory mutations appears to be to activate wild-type C-Raf, which then signals to ERK.

[0016] BRAF has also emerged as an important drug target for tumor therapy. Drugs that treat cancers driven by BRAF mutations have been developed. On August 17, 2011, one of them, vemurafenib, was approved by the FDA for the treatment of advanced-stage melanoma. Other BRAF-targeted kinase inhibitors include GDC-0879, PLX-4720, sorafenib tosylate, dabrafenib, and LGX818.

DDR2

[0017] Discoidin domain receptor family, member 2, also known as DDR2 or CD167b (cluster of differentiation 167b), is a receptor tyrosine kinase (RTK) that regulates cell growth, differentiation and metabolism in response to extracellular signals. DDR2 mutation has previously been reported in 3 to 4% of lung squamous cell carcinoma (SCC). In lung SCC, some cases with DDR2 mutation showed a clinical response to treatment with the tyrosine kinase inhibitor dasatinib (Cancer Discov. 2011 April 3; 1(1): 78-89). The data suggested that DDR2 may be an important therapeutic target in SCC.

[0018] DDR2 protein comprises an extracellular discoidin (DS) domain, a transmembrane domain and a kinase domain. The kinase domain is located at amino acids 563 to 849 of the full-length protein (which includes the signal peptide) and the DS domain is located at amino acids 22-399. The nucleotide sequence of human DDR2 mRNA variant 2 is shown in GenBank Accession no. NM_006182.

SUMMARY OF THE INVENTION

[0019] Methods for diagnosing melanoma in an individual are described. In one aspect of the present invention, a method for diagnosing melanoma in an individual comprises (a) analyzing a biological sample from the individual, (b) detecting the presence of a nucleic acid encoding a DDR2 protein with a mutation selected from the group consisting at R105C, P321L, R458H, S467F, P476S, I488S, F574C, S667F, S674F, R680L, L701F, R742Q, and T836A in the sample, and (c) diagnose the individual as having melanoma when the mutation is detected, thereby indicating that the individual has melanoma.

[0020] In another aspect, a method for diagnosing melanoma in an individual, comprises (a) analyzing a biological sample from the individual, (b) detecting the presence of a DDR2 protein with a mutation selected from the group consisting of R105C, P321L, R458H, S467F, P476S, I488S, F574C, S667F, S674F, R680L, L701F, R742Q, and T836A in the sample, and (c) diagnose the individual as having melanoma when the mutation is detected, thereby indicating that the individual has melanoma.

[0021] In particular embodiments, melanoma is advanced-stage melanoma. The individual may have a skin lesion and/or may be suspected of having a skin disorder such as, for example, skin cancer or melanoma.

[0022] Methods for diagnosing basal cell carcinoma in an individual are also described. In one aspect of the invention, a method for diagnosing basal cell carcinoma in an individual comprises: (a) analyzing a biological sample from the individual, (b) detecting the presence of a nucleic acid encoding a DDR2 protein with a selected mutation of the group consisting of N146K, R399Q, and S702F in the sample, and (c) diagnosing the individual as having basal cell carcinoma when the mutation is detected, thereby indicating that the individual has basal cell carcinoma.

[0023] In another aspect of the invention, a method for diagnosing basal cell carcinoma in an individual, comprises: (a) analyzing a biological sample from the individual, (b) detecting the presence of a DDR2 protein with a selected mutation of the group consisting of the N146K, R399Q, and S702F samples, and (c) diagnosing the individual as having basal cell carcinoma when the mutation is detected, thereby indicating that the individual has basal cell carcinoma.

[0024] The individual may have a skin lesion and/or may be suspected of having a skin disorder such as, for example, skin cancer, or basal cell carcinoma.

[0025] Methods for determining the probability that an individual with melanoma or basal cell carcinoma will respond to treatment with a kinase inhibitor are also described, comprising: (a) analyzing a biological sample from the individual, (b) detecting the presence of a DDR2 mutation that confers sensitivity to a kinase inhibitor in a DDR2 protein or nucleic acid in the sample, and (c) identify the individual as likely to respond to treatment with a kinase inhibitor when one or more of the DDR2 mutations is present , thereby indicating that the individual is likely to respond to treatment with a kinase inhibitor.

[0026] The DDR2 Mutation may be a mutation of the DDR2 protein selected from the group consisting of R105C, P321L, R458H, S467F, P476S, I488S, F574C, S667F, S674F, R680L, L701F, R742Q and T836A or a nucleic acid which encodes a DDR2 protein with a mutation selected from the group consisting of R105C, P321L, R458H, S467F, P476S, I488S, F574C, S667F, S674F, R680L, L701F, R742Q and T836A. The presence of any of these mutations may be present in an individual with advanced-stage melanoma.

[0027] The method may further comprise detecting the presence of a BRAF mutation such as V600E or V600K in the individual's BRAF.

[0028] In some embodiments, the DDR2 mutation is a mutation of the DDR2 protein selected from the group consisting of N146K, R399Q and S702F; or a DDR2 nucleotide sequence encoding a DDR2 mutation selected from the group consisting of N146K, R399Q and S702F. The presence of any of these mutations may be present in an individual with basal cell carcinoma.

[0029] In one embodiment, the step of analyzing a biological sample comprises sequencing the DDR2 gene for the presence of mutations known to confer sensitivity to DDR2 inhibitors. In some embodiments, the DDR2 nucleic acid sequence is examined for one or more mutations encoding N146K, R399Q, S702F, R105C, P321L, R458H, S467F, P476S, I488S, F574C, S667F, S674F, R680L, L701F, R742Q , or T836A in DDR2.

[0030] Methods for identifying an individual with melanoma or basal cell carcinoma as a candidate for therapy with a DDR2 inhibitor are also described. In some embodiments, a method for identifying an individual with melanoma or basal cell carcinoma as a candidate for therapy with a DDR2 inhibitor, comprises sequencing the DDR2 gene for the presence of mutations known to confer sensitivity to DDR2 kinase inhibitors. In some embodiments, the DDR2 sequence is examined for a sequence encoding at least one mutation selected from the group consisting of R105C, P321L, R458H, S467F, P476S, I488S, F574C, S667F, S674F, R680L, L701F, R742Q, and T836A. In an alternative method, a DDR2 inhibitor therapy candidate is identified by DDR2 expression levels, where low DDR2 expression levels such as DDR2 transcript levels relative to normalized GAPDH below 0.025 indicate that the individual is a candidate for therapy.

[0031] The invention comprises a method for treating melanoma or basal cell carcinoma in an individual comprising administering to the individual a therapeutically effective amount of a DDR2 inhibitor. In some embodiments, the melanoma is advanced-stage melanoma. Suitable DDR2 inhibitors include kinase inhibitors, siRNA, shRNA, and an antibody that specifically binds to DDR2 or to a DDR2 with at least one mutation selected from the group consisting of R105C, P321L, R458H, S467F, P476S, I488S, F574C, S667F , S674F, R680L, L701F, R742Q and T836A. In some embodiments, the DDR2 inhibitor is a tyrosine kinase inhibitor that inhibits the kinase activity of DDR2. In some embodiments, the kinase inhibitor inhibits the tyrosine kinase activity of DDR2 with at least one mutation in the kinase domain. In some embodiments, the kinase inhibitor inhibits the tyrosine kinase activity of DDR2 with at least one mutation in the discoidin (DS) domain. In some embodiments, the tyrosine kinase inhibitor inhibits the kinase activity of DDR2 with at least one mutation selected from the group consisting of R105C, P321L, R458H, S467F, P476S, I488S, F574C, S667F, S674F, R680L, L701F, R742Q and T836A.

[0032] Methods described herein can be used to treat melanoma or basal cell carcinoma in an individual with a DDR2 mutation selected from the group consisting of a mutation in the DDR2 discoidin domain, a mutation in the DDR2 intracellular interaction domain, and a mutation in the DDR2 kinase domain. DDR2 Mutation can be a germline mutation or a somatic cell mutation. In some embodiments the DDR2 mutation is an N146K, R399Q or S702F mutation. The DDR2 Mutation can be encoded by a mutated DDR2 gene.

[0033] With regard to therapy, the individual may be screened for mutations in the DDR2 protein, comprising sequencing a DDR2 nucleic acid from the individual to determine whether the nucleic acid encodes a DDR2 protein with a mutation and subsequently the probability that the individual will respond to therapy with a DDR2 inhibitor. In particular embodiments, the individual's DDR2 sequence may be determined prior to initiation of treatment or during treatment.

[0034] An individual with melanoma or basal cell carcinoma may harbor a mutation in a DDR2e nucleic acid sequence/or a mutation in a BRAF nucleic acid sequence. The mutation may be in the individual's DNA and/or genomic RNA. In some embodiments, the methods for treating melanoma or basal cell carcinoma described herein are performed in an individual who does not harbor a mutation in a DDR2 or a BRAF nucleic acid, or in an individual who carries a DDR2 and/or BRAF gene or Mutated RNA.

[0035] In another embodiment, an individual with melanoma or basal cell carcinoma is also treated with a BRAF inhibitor, such as an inhibitor that inhibits BRAF activity with a mutation at codon 600 (such as a V600E or V600K mutation). ) or other sensitive BRAF mutations in addition to being treated with a DDR2 inhibitor. Suitable BRAF inhibitors include BRAF kinase inhibitors such as, for example, vemurafenib, GDC-0879, PLX-4720, sorafenib tosylate, dabrafenib and LGX818.

[0036] Compositions for treating melanoma and/or basal cell carcinoma are also provided. In one embodiment, a composition for treating melanoma or basal cell carcinoma comprises a DDR2 inhibitor alone, or with a BRAF inhibitor. The DDR2 inhibitor may be a kinase inhibitor or one or more inhibitors selected from the group consisting of siRNA targeting a DDR2 nucleic acid, shRNA targeting a DDR2 nucleic acid, and an antibody that specifically binds to a DDR2 polypeptide. and inhibits the kinase activity of DDR2. In some embodiments, the DDR2 kinase inhibitor inhibits DDR2 with mutations in the kinase domain. In some embodiments, the inhibitor inhibits DDR2 with mutations in the discoidin domain. In some embodiments, the composition comprises a DDR2 kinase inhibitor that inhibits the kinase activity of DDR2 with one or more of the mutations R105C, P321L, R458H, S467F, P476S, I488S, F574C, S667F, S674F, R680L, L701F, R742Q and T836A. The BRAF inhibitor can be, for example, a kinase inhibitor such as vemurafenib, GDC-0879, PLX-4720, sorafenib tosylate, dabrafenib and LGX818.

[0037] The invention also includes kits. For example, a kit for identifying the presence of DDR2 and/or BRAF mutations, the kit comprising at least one primer oligonucleotide selected from

BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 is a sequence alignment of the DDR2 protein (top lines) compared to DDR1 (base) showing the conserved sequence between the two proteins at the sites of DDR2 mutations seen in DS extracellular domains (amino acids 22-399) and the kinase domain (amino acids 563 to 849) including F574C, S667F, R680L, L701F, R742Q, and T836A.

[0039] Figure 2 shows a sequence alignment of three mutations in the DDR2 kinase domain, F574C, S667F and L701F, identified by Ion Torrent sequencing of genomic DNA extracted from macrodissected formalin-fixed paraffin-embedded (FFPE) melanoma sections; middle panels show mutation confirmations by bidirectional Sanger sequencing; bottom panels show alignment of mutations in the DDR2 kinase domain with homologous locations in other kinases, including BRAF, EGFR, and ALK.

[0040] Figure 3 shows DDR2 expressed at low levels in melanoma mutated in DDR2.

[0041] Figure 4 shows a mutation of the S702F kinase domain in basal cell carcinoma.

[0042] Figure 5 shows biallelic mutations N146K and R399Q of DDR2 in basal cell carcinoma.

DETAILED DESCRIPTION Definitions

[0043] Certain terms used in this description have the following defined meanings. Terms that are not defined have their meanings recognized in the art. That is, unless otherwise defined, all technical and scientific terms used herein have the same meanings commonly understood by those skilled in the art to which this invention relates.

[0044] As used herein, unless otherwise established, the singular forms “a”, “an”, “the” and “a” also include the plural. Thus, for example, a reference to “an oligonucleotide” includes a plurality of oligonucleotide molecules, a reference to tag is a reference to one or more tags, a reference to probe is a reference to one or more probes, and a reference to “a nucleic acid” is a reference to one or more polynucleotides.

[0045] As used herein, unless otherwise indicated, with reference to a numerical value, the term “about” means plus or minus 10% of the enumerated value.

[0046] As used herein, the terms “isolated”, “purified” or “substantially purified” refer to molecules, such as nucleic acid, that are removed from their natural environment, isolated or separated, and are at least 60 % free, preferably 75% free, and more preferably 90% free from other components with which they are naturally associated. An isolated molecule is therefore a substantially purified molecule.

[0047] A “fragment” in the context of a gene fragment or a chromosome fragment refers to a sequence of nucleotide residues that is at least about 10 nucleotides long, at least about 20 nucleotides long, at least about 25 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 100 nucleotides, at least about 250 nucleotides, at least about 500 nucleotides, at least about 1,000 nucleotides , at least about 2,000 nucleotides.

[0048] The terms “identity” and “identical” refer to a degree of identity between sequences. There may be partial identity or complete identity. A partially identical sequence is one that is less than 100% identical to another sequence. Partially identical sequences may have an overall identity of at least 70% or at least 75%, at least 80% or at least 85%, or at least 90% or at least 95%.

[0049] The term “detectable tag” as used herein refers to a molecule or a compound or a group of molecules or a group of compounds associated with a probe and is used to identify the probe hybridized to a genomic nucleic acid or reference nucleic acid.

[0050] As used herein, the term “detect” refers to observing a signal from a detectable mark to indicate the presence of a target.

[0051] More specifically, detection is used in the context of detecting a specific sequence.

[0052] The term “multiple PCR” as used herein refers to an assay that provides simultaneous amplification and detection of two or more products in the same reaction vessel. Each product is primed using a distinct oligonucleotide primer pair. A multiple reaction may additionally include probes specific to each product that are detectably labeled with different detectable moieties.

[0053] The expression “nested polymerase chain reaction” is a modification of polymerase chain reaction that, in the present context, is performed to add sequences to an amplicon. Nested polymerase chain reaction involves two sets of oligonucleotide primers, used in two successive polymerase chain reaction runs, the second set designed to amplify the target of the first product run.

[0054] As used herein, the term “oligonucleotide” refers to a small polymer composed of deoxyribonucleotides, ribonucleotides, or any combination thereof. Oligonucleotides are generally between about 10, 11, 12, 13, 14, 15, 20, 25 or 30 to about 150 nucleotides (nt) in length, more preferably about 10, 11, 12, 13, 14, 15 , 20, 25 or 30 to about 70 nt.

[0055] As used herein, the term “subject” or “individual” refers to a mammal, such as a human, but can also be another animal such as a domestic animal (for example, a dog, cat or the like). ), a farm animal (e.g., a cow, a goat, a pig, a horse, or the like) or a laboratory animal (e.g., a monkey, a rat, a mouse, a rabbit, a guinea pig, -India, or similar).

[0056] The terms “complement”, “complementary” or “complementarity” as used herein with reference to polynucleotides (that is, a sequence of nucleotides such as an oligonucleotide or a genomic nucleic acid) related by base pairing rules. The complement of a nucleic acid sequence as used herein refers to an oligonucleotide that, when aligned with the nucleic acid sequence in such a manner that the 5' end of one sequence is paired with the 3' end of the other, is in “antiparallel association”. For example, the sequence 5‘-A-G-T-3‘ is complementary to the sequence 3‘-T-C-A-5‘. Certain bases not commonly found in natural nucleic acids may be included in the nucleic acids of the present invention and include, for example, inosine and 7-deazaguanine. Complementarity does not need to be perfect; Stable duplexes may contain imperfectly paired base pairs or unconjugated bases. Those skilled in the art of nucleic acid technology can determine duplex stability empirically by considering numerous variables including, for example, oligonucleotide length, oligonucleotide base composition and sequence, ionic strength, and incidence of mismatched base pairs. Complementarity can be “partial,” in which only some of the nucleic acid bases are conjugated according to base pairing rules. Or there may be “complete”, “total” or “entire” complementarity between the nucleic acids.

[0057] “Detection” of a mutation in a gene or protein can be carried out by carrying out an appropriate assay. To detect a mutation in a gene or protein in a biological sample, the biological sample is assayed to determine the presence or absence of the mutated gene or mutated protein. The assay may include extracting nucleic acid (such as, for example, total genomic DNA and/or RNA) from the sample and analyzing the extracted nucleic acid by methods known in the art. An assay may involve isolating the protein from the biological sample and analyzing the protein. However, an assay need not involve either nucleic acid extraction or protein isolation. In other words, some assays can be used that directly analyze a biological sample without extracting or isolating nucleic acid or protein.

Diagnostic Methods

[0058] In one embodiment, a method for diagnosing melanoma in an individual is described, comprising (a) analyzing a biological sample from the individual, (b) detecting the presence of a DDR2 protein with a mutation selected from the group consisting of R105C , P321L, R458H, S467F, P476S, I488S, F574C, S667F, S674F, R680L, L701F, R742Q, and T836A or a nucleic acid encoding a DDR2 protein such as that in the sample, and (c) diagnosing the individual as having melanoma when the mutation is detected, thereby indicating that the individual has melanoma. Melanoma may be a particular subset of melanoma. In some embodiments, the method is to diagnose advanced-stage melanoma.

[0059] Methods for diagnosing basal cell carcinoma in an individual are also described. A method for diagnosing basal cell carcinoma in an individual comprises (a) analyzing a biological sample from the individual, (b) detecting the presence of a DDR2 protein with a mutation selected from the group consisting of N146K, R399Q and S702F, or a DDR2 nucleic acid encoding the mutated DDR2 protein in the sample, and (c) identifying the individual with basal cell carcinoma when the mutation is present, thereby indicating that the individual has basal cell carcinoma.

[0060] The nucleic acid can be DNA and/or RNA.

[0061] Diagnostic methods can be performed on an individual with a skin lesion. A “skin lesion” is an area of variation in color and/or texture on the skin. Alternatively or additionally, the individual may be suspected of having a skin disorder such as, for example, skin cancer, melanoma or basal cell carcinoma.

Methods of Classifying/Predicting Response to Treatment

[0062] Another aspect of the present invention provides a method for determining the probability of response to treatment with a kinase inhibitor in an individual with melanoma or basal cell carcinoma, comprising: (a) analyzing a biological sample from the individual to detect the presence of a DDR2 mutation that confers sensitivity to a kinase inhibitor, and (b) identify the individual as likely to respond to treatment with a kinase inhibitor when one or more DDR2 mutations are present, thereby indicating that the individual probably responds to treatment with a kinase inhibitor. In some embodiments, the DDR2 mutation is an amino acid mutation in a DDR2 protein selected from the group consisting of R105C, P321L, R458H, S467F, P476S, I488S, F574C, S667F, S674F, R680L, L701F, R742Q, and T836A. In other embodiments, the DDR2 mutation is a nucleic acid sequence encoding a DDR2 protein with a mutation selected from the group consisting of R105C, P321L, R458H, S467F, P476S, I488S, F574C, S667F, S674F, R680L, L701F , R742Q and T836A.

[0063] In some embodiments, the DDR2 mutation is a mutation of the DDR2 protein selected from the group consisting of N146K, R399Q and S702F. In some embodiments, the DDR2 mutation is a DDR2 nucleic acid sequence that encodes a DDR2 mutation selected from the group consisting of N146K, R399Q, and S702F.

[0064] The method may additionally comprise detecting the presence of a V600E or V600K mutation in the individual's BRAF. The individual may have advanced-stage melanoma.

[0065] Yet another aspect of the present invention describes a method for stratifying melanoma into early and late stage, comprising (a) analyzing a biological sample from an individual with melanoma or suspected of having melanoma, (b) detecting the presence of a mutation of DDR2, such as R105C, P321L, R458H, S467F, P476S, I488S, F574C, S667F, S674F, R680L, L701F, R742Q or T836A in the sample, and (c) identify melanoma as a later stage when the DDR2 mutation is detected . The presence of these mutations is indicative of later stage melanoma given their absence in primary cutaneous melanomas and their presence in secondary cutaneous nodules or metastatic lesions that have an adverse prognosis (Balch CM et al. Prognostic Factors Analysis of 17,600 Melanoma Patients: Validation of the American Joint Committee on Cancer Melanoma Staging System. JCO August 15, 2001 vol. 19 no. 16 3622-3634, Balch CM et al. Final Version of 2009 AJCC Melanoma Staging and Classification. JCO December 20, 2009 vol. 27 no. 36 6199- 6206).

[0066] In one aspect of the present invention, an individual is identified as likely to respond to therapy with a DDR2 inhibitor such as, for example, a DDR2 kinase inhibitor, when relative DDR2 transcript levels are normalized by GAPDH in a biological sample from the individual are below 0.025.

[0067] The term “biological sample” as used herein refers to a sample containing a nucleic acid of interest. A biological sample may comprise a clinical sample (i.e., obtained directly from a patient) or isolated nucleic acids and may be cellular or acellular fluid and/or tissue samples (e.g., biopsy). In some embodiments, a sample is obtained from a tissue or bodily fluid collected from a subject. Sample sources include, but are not limited to, sputum (processed or unprocessed), bronchial alveolar lavage (BAL), type (e.g., lymphocytes), body fluids, cerebrospinal fluid (CSF), urine, bronchial lavage (BW) , whole blood cells or blood isolated from any plasma, serum or tissue (e.g. biopsy material). Methods of obtaining test samples and reference samples are well known to those skilled in the art and include, but are not limited to, aspirations, tissue sections, collection of blood or other fluids, surgical or needle biopsies, collection of paraffin-embedded tissue, collection of bodily fluids, collection of feces and similar. In the present context the biological sample is preferably blood, serum or plasma. The term "patient sample" as used herein refers to a sample obtained from a search diagnosis and/or treatment of a human disease, especially prostate disease.

[0068] To detect the presence of a DDR2 or BRAF mutation, nucleic acid samples or target nucleic acids can be amplified and sequenced by various methods known to those skilled in the art including Sanger sequencing and so-called Next Generation Sequencing (NGS). ). Next run sequencing lowers costs and greatly increases speed over industry standard dye-terminator methods. Examples of NGS include: (a) Massively Parallel Signature Sequencing (MPSS) (b) Polony Sequencing combined with a paired-label library in vitro with emulsion PCR (c) 454 Pyrosequencing (d) Solexa Sequencing (e) Technology SOLiD (f) DNA Nanosphere (g) Single Molecule Heliscope (h) Single Molecule Real Time (SMRT) and (i) Ionic Semiconductor Sequencing

[0069] Ionic semiconductor sequencing couples standard sequencing chemistry with a novel semiconductor-based detection system that detects hydrogen ions that are released during DNA polymerization, unlike optical methods used in other sequencing systems. A microwell containing a template DNA strand to be sequenced is flooded with a single type of nucleotide. If the introduced nucleotide is complementary to the leading template nucleotide, it is incorporated into the growing complementary strand. This causes the release of a hydrogen ion that triggers a hypersensitive ionic sensor, which indicates that a reaction has occurred. If homopolymer repeats were present in the template sequence, multiple nucleotides will be incorporated in a single cycle. This leads to a corresponding number of hydrogens released and a proportionally larger electronic signal.

[0070] The terms “amplification” or “amplify” as used herein include methods for copying a target nucleic acid, thereby increasing the number of copies of a selected nucleic acid sequence. Amplification can be exponential or linear. A target nucleic acid can be either DNA or RNA. Sequences amplified in this way form an “amplification product”, also known as an “amplicon”. Although the exemplary methods described below relate to amplification using the polymerase chain reaction (PCR), numerous other methods are known in the art for amplification of nucleic acids (e.g., isothermal methods, rolling circle methods, etc.). ). Those versed in the technique will understand that these other methods can be used both in place of PCR methods and alongside them. see, for example, Saiki, “Amplification of Genomic DNA” in PCR Protocols, Innis et al., Eds., Academic Press, San Diego, CA 1990, pp. 13-20; Wharam et al., Nucleic Acids Res., 29(11):E54-E54, 2001; Hafner et al., Biotechniques, 30(4):852-56, 858, 860, 2001; Zhong et al., Biotechniques, 30(4):852-6, 858, 860, 2001.

[0071] A key feature of PCR is “thermocycling” which, in the present context, comprises repeated cycling through at least three different temperatures: (1) melting/denaturation, typically at 95°C (2) annealing of a primer oligonucleotide into the Target DNA at a temperature determined by the melting point (Tm) of the region of homology between the primer oligonucleotide and the target and (3) extension at a polymerase-dependent temperature, most commonly 72°C. These three temperatures are then repeated numerous times. Thermocycling protocols typically also include an extended first denaturation period, and end in an extended extension period.

[0072] The Tm of a primer oligonucleotide varies according to the length, G+C content and buffer conditions, among other factors. As used herein, Tm refers to that in the buffer used for the reaction of interest.

[0073] An oligonucleotide (e.g., a probe or a primer oligonucleotide) that is specific for a target nucleic acid will “hybridize” to the target nucleic acid under suitable conditions. As used herein, "hybridization" or "hybridizing" refers to the process by which a single strand of oligonucleotide anneals with a complementary strand through base pairing under defined hybridization conditions. It is a specific, that is, non-random, interaction between two complementary polynucleotides. Hybridization and hybridization resistance (i.e., the strength of association between nucleic acids) are influenced by factors such as the degree of complementarity between the nucleic acids, stringency of the conditions involved, and the Tm of the hybrid formed.

Treatment Methods

[0074] In one aspect of the invention, a method of treating melanoma or basal cell carcinoma in an individual comprises administering to the individual a therapeutically effective amount of a DDR2 inhibitor. A “DDR2 inhibitor” is a compound that inhibits DDR2 function (including DDR2 kinase activity) and includes kinase inhibitors that inhibit DDR2 kinase activity as well as siRNA specific for DDR2 nucleic acid, shRNA specific for DDR2 nucleic acid, and an antibody that binds to DDR2 and inhibits the associated DDR2 kinase activity.

[0075] An "antibody" includes a polyclonal antibody, a monoclonal antibody, antigen-binding fragments thereof such as F(ab').sub.2 and Fab fragments, and a single-chain antibody.

[0076] As used herein, a “kinase inhibitor” is a composition that inhibits the kinase activity of a protein kinase. A “tyrosine kinase inhibitor” is a composition that inhibits the tyrosine kinase activity of a protein tyrosine kinase, and a “serine/threonine kinase inhibitor” is a compound that inhibits the serine/threonine kinase activity of a serine/threonine protein kinase . Exemplary kinase inhibitors include imatinib, nilotinib, dasatinib, GDC-0879, PLX-4720, sorafenib, tosylate, dabrafenib, vemurafenib, and LGX818. Imatinib mesylate (also briefly known as STI571 or 2-phenylaminopyrimidine or "imantinib"; marketed as a drug under the brand name "Gleevec" or "Glivec") is an ATP-competitive inhibitor of tyrosine kinase activity. Other kinase inhibitor drugs include bosutinib (SKI-606) and the aurora kinase inhibitor VX-680.

[0077] A “DDR2 kinase inhibitor” is a compound that inhibits the kinase activity of DDR2, including kinase activity of a DDR2 with at least one mutation selected from the group consisting of R105C, P321L, R458H, S467F, P476S, I488S , F574C, S667F, S674F, R680L, L701F, R742Q, T836A, N146K, R399Q and S702F. A DDR2 kinase inhibitor can inhibit kinase activity of a DDR2 protein directly, or it can act upstream to inhibit DDR2 nucleic acid (e.g., inhibiting transcription or translation). Exemplary DDR2 kinase inhibitors include imatinib, nilotinib, and dasatinib.

[0078] In one embodiment of this aspect of the invention, the method comprises administering to the individual a therapeutically effective amount of a BRAF inhibitor, in addition to the DDR2 inhibitor. A “BRAF inhibitor” is any compound that inhibits BRAF function (including BRAF kinase activity) and includes kinase inhibitors that inhibit BRAF kinase activity, as well as BRAF nucleic acid-specific siRNA, nucleic acid-specific shRNA of BRAF and antibodies that bind to BRAF and inhibit associated BRAF kinase activity. The BRAF inhibitor may be a serine/threonine kinase inhibitor. A “BRAF kinase inhibitor” is a compound that inhibits the kinase activity of BRAF. Exemplary BRAF kinase inhibitors include GDC-0879, PLX-4720, sorafenib, vemurafenib, tosylate, dabrafenib and LGX818.

[0079] Routes and frequency of administration of the therapeutic agents described here, as well as dosage, will vary from individual to individual as well as with the drug selected, and can be easily established using standard techniques. In general, pharmaceutical compositions can be administered by injection (e.g., intracutaneous, intratumoral, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration) or orally. In a particular embodiment, the pharmaceutical composition is administered orally. In one example, between 1 and 10 doses may be administered over a period of 52 weeks. Preferably, 6 doses are administered at 1 month intervals, and booster treatments may be given periodically thereafter. Alternative protocols may be appropriate for individual patients. In one embodiment, 2 intradermal injections of the composition are administered at 10 day intervals.

[0080] A "solid oral dosage form", "oral dosage form", "unit dose form", "dosage form for oral administration" and the like are used interchangeably, and refer to a pharmaceutical composition in the form of a tablet, capsule, capsule-shaped tablet, gelatin capsule, gelatin tablet, pill and the like.

[0081] Dosage forms typically include an "excipient", which as used herein, is any component of a dosage form that is not an API. Excipients include binders, lubricants, diluents, disintegrants, coatings, barrier layer components , glidants, and other components. Excipients are known in the art (see HANDBOOK OF PHARMACEUTICAL EXCIPIENTS, FIFTH EDITION, 2005, edited by Rowe et al., McGraw Hill). Some excipients serve multiple functions or are so-called high functionality excipients. For example, talc can act as a lubricant, and a non-stick, and a glidant. See Pifferi et al., 2005, "Quality and functionality of excipients" Farmaco. 54:1-14; and Zeleznik and Renak, Business Briefing: Pharmagenerics 2004.

[0082] An adequate dose is an amount of a compound that, when administered in the manner described above, is capable of promoting an anti-cancer immune response, and is at least 10 to 50% above the basal (i.e., untreated) level. Such a response can be monitored using conventional methods. In general, for pharmaceutical compositions, the amount of each drug present in a dose ranges from about 100 μg to 5 mg per kg of host, but those skilled in the art will appreciate that specific doses depend on the drug being administered and are not necessarily limited to this general range. Likewise, adequate volumes for each administration will vary with the size of the patient.

[0083] In the context of treatment, a "therapeutically effective amount" of a drug is a pharmaceutically acceptable amount of salt that eliminates, alleviates, or provides relief from the symptoms for which it is administered. The described compositions are administered in any suitable manner, often with pharmaceutically acceptable carriers. Suitable methods of administering treatment in the context of the present invention to a subject, and although more than one route may be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route. The dose administered to a patient, in the context of the present invention, could be sufficient to promote a beneficial therapeutic response in the patient over time, or inhibit disease progression. Thus, the composition is administered to a subject in an amount sufficient to elicit an effective response and/or to alleviate, reduce, cure or at least partially prevent symptoms and/or complications of the disease. An amount adequate to accomplish this is defined as a "therapeutically effective dose."

[0084] In general, an appropriate dosage and treatment regimen involves administration of the active compound(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit. A response like this can be monitored by establishing a better clinical outcome (e.g., more frequent remissions, complete, partial, or longer disease-free survival) in treated patients compared with untreated patients.

[0085] In some embodiment, IC50 for inhibition of wild-type DDR2 kinase activity is in the range of 600 nM for imatinib, 50 nM for nilotinib and 1.5 nM for dasatinib (Day E, et al. Inhibition of activation 1 and 2 of the collagen-induced discoidin domain receptor by imatinib, nilotinib, and dasatinib. Molec Cell Pharm 2008;599:44-53), which are below the low plasma concentrations obtainable from the drugs at their current daily oral dosage (4 uM, 2 uM and 100 nM respectively, see Bradeen et al. Blood 2006;108:2332-2338).

[0086] The DDR2 inhibitor and BRAF inhibitor, if both administered, can be administered sequentially or simultaneously, and can be formulated separately or as a single composition.

Kits

[0087] In one embodiment of the invention, a kit can be used to conduct the diagnostic and prognostic methods described here. Typically, the kit would contain, in a compartmentalized vehicle or container, reagents used in any of the above-described modalities of the diagnostic method. The vehicle may be a container or support, in the form of, for example, a bag, box, tube, shelf, and is optionally compartmentalized. The vehicle can define a closed confinement for security purposes during transport and storage. A kit described here may contain printed or electronic instructions for performing an assay employing the reagents contained in the kit. In one embodiment, the kit includes one or more PCR primer oligonucleotides capable of amplification and sequencing. Relevant oligonucleotide primers include

[0088] As used herein, a “primer oligonucleotide” is an oligonucleotide that is complementary to a target nucleotide sequence and leads to the addition of nucleotides at the 3' end of the primer oligonucleotide in the presence of a DNA or RNA polymerase. The 3' nucleotide of the primer oligonucleotide could generally be identical to the target sequence at a corresponding nucleotide position for optimal extension and/or amplification. The term “primer oligonucleotide” includes all forms of primer oligonucleotides that can be synthesized, including peptide nucleic acid primer oligonucleotides, locked nucleic acid primer oligonucleotides, phosphorothioate modified primer oligonucleotides, labeled primer oligonucleotides, and the like. As used herein, a “forward oligonucleotide primer” is a primer oligonucleotide that is complementary to the antisense DNA strand. A “reverse oligonucleotide primer” is complementary to the sense strand of DNA.

[0089] The kit may also include suitable buffers, reagents for isolating nucleic acid and instructions for use. Kits may also include a microarray to measure miRNA level.

[0090] The primer oligonucleotides can be labeled with a detectable label such as radioactive isotopes, or fluorescence labels. Instructions for using the kit or reagents contained therein are also included in the kit.

EXAMPLES Example 1: Identification of novel DDR2 mutations in cancer

[0091] The following test was conducted, which reports a DETECTED/NOT DETECTED result for any mutations or insertions-deletions observed in the sixteen DDR2 coding exons and in BRAF exons 11 and 15, which are the sites of >99% of BRAF mutations of known activation in identified melanoma.

[0092] Total nucleic acid was extracted from paraffin-embedded tissues in block forms or affixed to slides. For a detection method, thirty-six pairs of oligonucleotide primers were designed to amplify full coding DDR2 and BRAF exon 11/15 sequences that are located on chromosome 1 and 7 in a microfluidic device, Access Array 48.48 chip. All amplicons from each sample were collected on the Access Array chip. The pooled amplicons were end repaired and a unique bar-coded adapter A and a P1 adapter were ligated into both amplicon ends for each sample. The resulting DNA amplicons were the 'DNA sequencing library' with a unique barcode sequence for each sample. 12~16 DNA libraries were mixed equally to produce a 300 million DNA fragment pool. Emulsion PCR was performed by mixing 1 mL of PCR reaction mixture containing the library pool and Ionic Sphere Particle (ISP) and 9 mL of oil. ISP microspheres were recovered from emulsion PCR and template-positive ISPs were enriched, the ISPs were loaded onto a chip for semiconductor sequencing analysis. Raw sequencing data was transferred to Ion Torrent and processed into next generation standard sequence formats. Sequence data were aligned and analyzed by Ion Suite Software, SeqNext or NEXTGen software using GenBank accession number NM__006182 as reference. This semi-quantitative test reports a DETECTED/NOT DETECTED result for any mutations or insertions-deletions observed in the sixteen coding DDR2 exons and in BRAF exons 11 and 15, which are the sites of >99% of known activating BRAF mutations in identified melanoma. Table 1 Controls Used

Example 2

[0093] Somatic mutations in DDR2 were identified in melanomas that contained a V600 BRAF mutation. All mutations were detected by advanced Ion Torrent sequencing and then confirmed by bidirectional Sanger sequencing. Table 2. Unpublished DDR2 Somatic Mutations Identified in Melanoma.

[0094] Unpublished DDR2 mutations in conserved residues in DDR2 including R105C, P321L, R458H, F574C, S667F and L701F were identified in human malignant melanoma. Approximately 50% of DDR2 mutations in melanoma have been associated with simultaneous BRAF serine/threonine kinase mutations at codon 600. Based on mutation levels, DDR2 and BRAF mutations are predicted to be present in most, if not all, cells. tumors.

[0095] These findings suggest that dysregulation by a DDR2 mutation plays a role in melanoma progression because, unlike BRAF mutations, we did not observe DDR2 mutations in moles. This finding is interesting in light of a previous study demonstrating that downregulation of DDR2 in a melanoma cell line can modulate its metastatic potential (Oncol Rep 2011 Oct;26(4):971-8) and mediate cell cycle arrest and the adhesion phenotype of primary tumor cells (Frontiers in Bioscience 10, 2922-2931, September 1, 2005). This finding could also be used to distinguish melanoma from moles, which can be difficult to distinguish both clinically and histologically.

[0096] DDR2 mutations provide a targetable genetic characteristic in a group of melanomas for tyrosine kinase inhibitors such as imatinib, dasatinib and nilotinib. IC50 for inhibition of wild-type DDR2 kinase activity is in the range of 600 nM for imatinib, 50 nM for nilotinib, and 1.5 nM for dasatinib (Day E, et al. Inhibition of induced discoidin domain receptor activation 1 and 2 by collagen by imatinib, nilotinib and dasatinib. Molec Cell Pharm 2008; 599:44-53), which are below achievable plasma concentrations of the drugs at their current daily oral dosage (4 uM, 2 uM and 100 nM respectively, see Bradeen et al. Blood 2006;108:2332-2338).

[0097] Therefore, melanoma can be treated by targeting the kinase activity of DDR2 together with targeting the kinase activity of BRAF. This discovery therefore opens up an additional therapeutic option for melanoma patients.

Example 3

[0098] Genomic DNA extracted from FFPE blocks or tissue sections in routine clinical tumor samples can be sequenced by standard PCR-based dideoxy chain termination sequencing ("Sanger" method). Relevant oligonucleotide primers include: Table 3. DDR2 PCR/Sequencing Oligonucleotide Primers

Example 4

[0099] To assess whether DDR2 is mutated or dysregulated in melanoma, we classified DNA extracted from a variety of different melanocytic lesions, focusing particularly on high-stage melanomas that would be candidates for kinase inhibitor therapy.

[00100] Mutations in the DDR2 kinase domain, F574C, S667F and L701F, were identified by Ion Torrent sequencing of genomic DNA extracted from macrodissected FFPE sections of primary melanomas. Normalized to pathologist estimates of percentages of tumor in the macrodissected areas, all mutations were predicted to be present at heterozygous levels (percentages indicate non-normalized reads of mutant sequence compared to wild type). Mutations were confirmed by bidirectional Sanger sequencing.

[00101] Ion sequencing was performed on the PGM platform on a 316 chip and 200 base sequencing chemistry; Emulsion PCR was performed on the OneTouch ES or 2 (all from Life Technologies). To produce the library, singleplex PCR was performed on the Access Arrangement system (Fluidigm, São Francisco do Sul, CA) using custom-designed oligonucleotide primers for 48 amplicons covering the entire coding region of DDR2 and exons 11 and 15 of BRAF. Sequence analysis was performed on SequencePilot software (JSI Medical Systems, Costa Mesa, CA) Sanger sequencing was performed on a 3700 Genetic Analyzer, following standard PCR, cleanup, and cycle sequencing methods using Big Dye v.3.1 reagents (Applied Biosystems , Foster City, CA)

[00102] Using a DNA-based Ion Torrent™ sequencing assay (Life Technologies, South San Francisco, CA) to evaluate the entire coding region of DDR2 as well as exons 11 and 15 of BRAF, with confirmation by the Sanger sequencing method, we identified erroneous DDR2 point mutations in 12/269 (4.5%) of melanoma cases. The mutation frequency did not differ notably by BRAF V600 mutation status in that DDR2 mutations were detected in 6/140 BRAF-mutated cases (V600E in 4, V600K in 2) and 7/129 without BRAF mutations. All but one of the DDR2-mutated melanomas were at an advanced stage: 3 were detected in extracutaneous metastases, and 7 in secondary skin or subcutaneous nodules, with no DDR2 mutations identified in primary cutaneous melanomas at the initial stage 31 classified. Also, no DDR2 mutations were identified in 29 benign moles, including blue moles, typical dermal and composite moles, and dysplastic moles.

[00103] In five cases, DDR2 mutations involved highly evolutionarily conserved residues in the kinase domain (e.g., F574C, S667F, and L701F shown in Figure 2) suggesting hypofunctional effects on DDR2 kinase activity. Mutations in the four other cases were R458H, S467F, P476S and I488S, all located in the DDR2-specific region of the cytoplasmic domain. The clustering of mutations in highly conserved kinase domain residues in melanoma was different from previous findings in lung carcinomas, where mutations were more widely dispersed and involved the discoidin and cytoplasmic domains and less conserved areas of the kinase domains.

[00104] DDR2 transcript levels were assessed from total RNA extracted from FFPE sections of macrodissected human melanoma samples using one-step cDNA/reverse transcription synthesis and real-time PCR with oligonucleotide assay primers/sets Gene Expression probe for the DDR1, DDR2, and GAPDH genes on the 7500 detection system (all from Applied Biosystems). DDR2 transcript levels were normalized to GAPDH transcripts using the delta Ct method. Samples include advanced-stage melanomas with DDR2 mutation (n = 5), without DDR2 or V600 BRAF mutation (wt, n = 17), and with V600 BRAF mutation but without DDR2 mutation (n = 20).

[00105] Using reverse transcription PCR on macrodissected FFPE melanoma samples, we noted that DDR2 was underexpressed in 4 of the 5 DDR2-mutated melanomas with available material compared to only a small minority of advanced-stage DDR2/BRAF-unmutated melanomas or cases of BRAF V600-mutated individuals who did not have DDR2 mutations (Figure 3). DDR2 mutations were observed in 4/10 cases with respect to DDR2/GAPDH transcript levels below 0.025 compared to 1/32 cases with ratios above 0.025 (p = .008). DDR1 transcript levels were more variable in the melanoma samples, but also low in the DDR2-mutated subgroup (data not shown). This suggests that DDR2 expression can be used as a classification tool for DDR2 mutations.

[00106] Downregulation of DDR2 has also been noted in lung carcinoma, with upregulation observed in some other tumor types. In the DDR2-mutated melanomas identified here, lower DDR2 activity could be produced by a combination of inactivating or hypofunctional mutations in one allele and transcriptional downregulation of the other allele.

[00107] Mutations of the kinase domain of DDR2 occur in a subset of BRAF-mutated advanced-stage melanomas and likely produce hypofunctional kinases based on the affected codons. In many cases, the DDR2-mutated overlaps with the BRAF-mutated group of typical sun-exposed melanomas, but, unlike BRAF, DDR2 mutations have not yet been observed in moles.

Example 5

[00108] Somatic DDR2 mutations have been identified in basal cell carcinoma, including in the discoidin domain (N146K), the intracellular interaction domain (R399Q) and the kinase domain (S702F). See Figures 4 and 5. The frequency of DDR2 mutations in a small sample of basal cell carcinomas was 32%.

[00109] It should be understood that although the present invention has been specifically described by preferred embodiments and optional features, modification, improvement and variation of the inventions incorporated therein described herein may be used by those skilled in the art, and that such modifications, improvements and variations are considered within the scope of this invention. The materials, methods, and examples provided here are representative of preferred embodiments, are exemplary, and are not intended to limit the scope of the invention.

[00110] The invention has been broadly and generically described here. Each of the narrower species and subgeneric groupings that fall within the generic description also forms part of the invention. This includes the generic description of the invention with a negative condition or limitation removing any matter in question from the genre, regardless of whether or not the excised material is specifically cited here.

[00111] Furthermore, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will appreciate that the invention is hereby also described in terms of any individual member or subgroup of members of the Markush group.

[00112] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the extent that each would be incorporated by reference individually. In case of conflict, this specification, including definitions, will prevail.

[00113] The inventions illustratively described here may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically described here. Thus, for example, the terms “comprising”, “including”, containing”, etc. should be read extensively and without limitation. Additionally, the terms and expressions employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is understood that various Modifications are possible within the scope of the claimed invention.

[00114] Other modalities are presented in the following claims.

Claims (5)

1. Method for diagnosing melanoma in an individual, characterized by the fact that it comprises: (a) amplifying, from genomic DNA extracted from a tissue sample of the individual suspected of having melanoma, the nucleic acid encoding a receptor 2 protein of the discoidin domain (DDR2) of SEQ ID NO: 170, (b) sequencing the amplicons obtained to detect the presence of a nucleic acid encoding the DDR2 protein with a mutation, wherein the mutation is selected from the group consisting of R105C, P321L, R458H, S467F, P476S, I488S, F574C, S667F, S674F, R680L, L701F, R742Q, and T836A, and (c) diagnose the individual as having melanoma when the DDR2 mutation is detected, thereby indicating that the individual has melanoma. 2. Method according to claim 1, characterized by the fact that the melanoma is advanced-stage melanoma. 3. Method for determining probability of responding to treatment with a kinase inhibitor in an individual with melanoma, characterized by the fact that it comprises: (a) amplifying, from genomic DNA extracted from a tissue sample of the individual suspected of having melanoma , the nucleic acid encoding a discoidin domain receptor 2 (DDR2) protein of SEQ ID NO: 170, (b) sequence the amplicons obtained to detect the presence of a nucleic acid sequence encoding the DDR2 protein having a selected mutation of the group consisting of R105C, P321L, R458H, S467F, P476S, I488S, F574C, S667F, S674F, R680L, L701F, R742Q, and T836A, and (c) identify the individual as likely to respond to treatment with a kinase inhibitor when a or more of the DDR2 mutations are detected, thereby indicating that the individual is likely to respond to treatment with a kinase inhibitor. 4. Method according to claim 3, characterized by the fact that it further comprises detecting the presence of a B-Raf proto-oncogene (BRAF) mutation, wherein the mutation is V600E or a V600K. 5. Method according to claim 3, characterized by the fact that the individual has advanced-stage melanoma.